Jason W. Johnston

Cryopreservation induces temporal DNA methylation epigenetic changes and differential transcriptional activity in Ribes germplasm.

The physiological and molecular mechanisms associated with acclimation and survival have been examined in four Ribes genotypes displaying differential cryotolerance. Changes in DNA methylation, nucleic acid and nucleoside composition were determined during acclimation and recovery of in vitro shoot-meristems from cryopreservation. DNA methylation was induced in the tolerant genotype, while demethylation was evident in sensitive genotypes. This response initially occurred during sucrose simulated acclimation, with progressive changes as shoots recovered from successive stages of the encapsulation-dehydration protocol. These methylation patterns existed in the initial vegetative cycle but regressed to control values following subculture, indicating the changes in DNA methylation to be a reversible epigenetic mechanism. RNA levels indicating transcriptional activity during the acclimation of nodal tissue are inversely linked to methylation changes, where activity appears to be up-regulated in the cryosensitive genotypes. Conversely, cryopreserved shoots show increased levels of both RNA and DNA methylation in the cryotolerant genotypes. Other nucleosides show post-transcriptional activity corresponds with tolerance during acclimation and cryopreservation. These observations connect physiological attributes to differential molecular changes in Ribes, the implications of which are discussed in relation to cryopreservation-induced apoptosis and genetic stability.

Exploring the physiological basis of cryopreservation success and failure in clonally propagated in vitro crop plant germplasm

An appraisal of potato and Ribes shoot meristem cryopreservation shows physiological factors influence survival and development, sometimes independently of protocol and genotype. Markers for oxidative damage incurred by cryostorage reveal two responses: (1) oxidative stress with an eventual decline in regrowth and (2) an oxidative burst associated with higher survival. Differential responses to cryoinjury are discussed in relation to in vitro ageing and genetic stability within the conceptual framework of cryobionomics. The possibility that cryopreservation-induced cell death and apoptosis occurs in plants is considered with respect to current concepts of animal cell cryoinjury. It is proposed that a more holistic approach is now required to understand the basis for success or failure of cryopreserved plant germplasm.;

Effects of osmotic pretreatments on oxidative stress, antioxidant profiles and cryopreservation of olive somatic embryos

A three-day pretreatment of olive somatic embryos (SE) with 0.75 M sucrose, combined with cryoprotection (0.5M DMSO, 1M sucrose, 0.5M glycerol and 0.009 M proline) and controlled rate cooling, supported regrowth (as 34.6% fresh weight gain) and resumption of embryo development after cryopreservation. Pretreatment with mannitol or sorbitol did not support regrowth. Profiles of sugars, proline, antioxidant enzymes, Reactive oxygen species (ROS), secondary oxidation products and ethylene were constructed for the most successful (0.75 M) pretreatment series. Sucrose was the optimal pretreatment for supporting recovery, it also elevated glutathione reductase (GR) activity compared to controls, whereas superoxide dismutase (SOD), catalase and guaiacol peroxidase activities remained relatively unchanged. Superoxide dismutase activity was higher in SE pretreated with sucrose, compared with those pretreated with polyols; H(2)O(2) was enhanced in SE pretreated with sorbitol and sucrose compared to mannitol. The overall trend for ethylene and OH production revealed their levels were highest in SE pretreated with polyols albeit, for individual treatments this was not always the case. Generally, pretreatments did not significantly change embryo secondary oxidation profiles of ThioBarbituric Acid Reactive Substances (TBARS) and Schiffs bases. In combination these studies suggest oxidative processes may influence regrowth of cryopreserved olive SE and that optimal pretreatments could, in part, increase tolerance by an overall enhancement of endogenous antioxidants (particularly GR), proline and sugars.

Headspace volatile markers for sensitivity of cocoa (Theobroma cacao L.) somatic embryos to cryopreservation

The mechanisms that reduce the viability of plant somatic embryos following cryopreservation are not known. The objective of the present study was to evaluate the sensitivity of cocoa (Theobroma cacao L.) somatic embryos at different stages of an encapsulation–dehydration protocol using stress-related volatile hydrocarbons as markers of injury and recovery. The plant stress hormone ethylene and volatile hydrocarbons derived from hydroxyl radicals (methane) and lipid peroxidation (ethane) were determined using gas chromatography headspace analysis. Ethylene and methane were the only volatiles detected, with both being produced after each step of the cryogenic protocol. Ethylene production was significantly reduced following exposure to liquid nitrogen, but then increased in parallel with embryo recovery. In contrast, the production of methane was cyclic during recovery, with the first cycle occurring earlier for embryos recovered from liquid nitrogen and desiccation than those recovered from earlier steps in the protocol. These results suggest that loss of somatic embryo viability during cryopreservation may be related to the oxidative status of the tissue, and its capacity to produce ethylene. This study has demonstrated that headspace volatile analysis provides a robust non-destructive analytical approach for assessing the survival and recovery of plant somatic embryos following cryopreservation.

Physical And Engineering Perspectives Of In Vitro Plant Cryopreservation

Cryopreservation is the conservation of living cells and organisms at ultra low temperatures usually at -196 o C in liquid nitrogen, it is a safe, long-term means of securing in vitro germplasm in culture collections. Cryogenic storage is used extensively in medical, horticultural, agricultural, aquaculture and forestry sectors and assists environmental research by preserving test organisms used in environmental monitoring. Designing and operating instruments and analytical equipment required to study and conserve biological samples at cryogenic temperatures poses a challenge dictated by the physical and safety constraints of operating at very low temperatures. This chapter overviews the physical and engineering aspects of in vitro plant cryopreservation including an introduction to the safe use of cryogenic systems. It concludes with a comparative case study of how thermal instrumentation may be applied to develop cryopreservation methods for temperate and tropical plant germplasm maintained in tissue culture. Cryobiology is a broad discipline and includes the preservation of a broad spectrum of biodiversity, as well as medical, polar and environmental low temperature research [1]. Plant cell cryopreservation requires the input of theoretical and practical expertise from diverse disciplines, including: engineering, computing and physics; chemistry, biology and biotechnology and, increasingly environmental knowledge. Cryobiologists network across multidisciplinary sectors, (www.cobra.ac.uk; http://www.sltb.info; http://www.societyforcryobiology.org/; http://www.agr.kuleuven.ac.be/dtp/tro/CRYMC EPT) which historically has lead to the development of generic analytical cryogenic instrumentation and storage technologies [1]. Cryogenic engineering is highly specialist, encompassing the design, construction and utilization of equipment capable of effective and safe operations at ultra low temperatures. In many situations this involves withstanding the physical constraints of operating, often rapidly, through different thermal cycles, under elevated pressures and in contact with liquid nitrogen vapour and